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Phos-Tag Acrylamide Mobility Shift Detection of Phosphorylated Proteins - Phosphate Affinity SDS-PAGE using Acrylamide-pendant Phos-tag TM - Ver. 8 (2010/8) 1. Introduction Phosphorylation is a fundamental covalent post-translational modification that regulates the function, localization, and binding specificity of target proteins. Methods for determining the phosphorylation status of proteins ( i.e., phosphoproteomics) are thus very important with respect to the evaluation of diverse biological and pathological processes. In 2002, Prof. Koike's group (Hiroshima University) reported that a dinuclear metal complex ( i.e. , 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) acts TM as a selective phosphate-binding tag molecule, Phos-tag in an aqueous solution at a neutral pH (e.g. , Kd 2- = 25 nM for phenyl phosphate dianion, Ph -OPO 3 ). Since then, various methods for phosphoproteome research have been developed using Phos-tag TM derivatives. Here, we demonstrate a novel application for detection of phosphorylated proteins in SDS-PAGE using an analogous Phos-tag TM complex with two manganese(II) ions, Mn 2+ –Phos-tag TM (a dinuclear manganese complex of acrylamide-pendant Phos-tag TM ligand). 2. Description of Acrylamide-pendant Phos-tag TM The acrylamide-pendant Phos-tag TM ligand (Phos-tag TM AAL-107) provides a phosphate affinity SDS-PAGE for mobility shift detection of phosphorylated proteins. This method requires only a general mini-slab PAGE system. The product is supplied as light yellow viscous oil (each at 10 mg in an airtight plastic tube), which has no irritant effect on the skin. Below 4˚C, the product is stable for at least 1 year. O NN N H OH HN O N N Phos-tagTM AAL-107 Mol. Wt.: 595 N N 3. Warning and Limitations Phos-tag TM AAL-107 is not for use in human diagnostic and the therapeutic procedures. Do not use internally or externally in human or animals. It's used only for research. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. 4. Advantages of Phos-tag TM SDS-PAGE # Radioactive and chemical labels are avoided. # Phosphoprotein isotypes can be detected as multiple migration bands in the same lane. # The procedure is almost the same as that for the general SDS-PAGE. # The binding specificity of Phos-tag TM is independent on amino acid and sequence context. # Downstream procedures such as Western blot analysis and MS analysis are applicable. # Phos-tag TM AAL-107 dissolved in distilled water is stable for at least 3 months. # The time-course ratio of phosphorylated and non-phosphorylated proteins can be determined. # Separation of phosphoprotein isotypes having the same number of phosphate groups is possible. - 1 - 5. Principle of Mn 2+ –Phos-tag TM SDS-PAGE 6. Solutions for Phos-tag TM SDS-PAGE Sol. A : 30% w/v acrylamide solution (100 mL, stored at ca. 4˚C in the dark) # acrylamide 29.0 g # N,N' -methylene-bisacrylamide 1.0 g # distilled water for preparation of the 100 mL solution a proper quantity Sol. B : 1.5 mol/L Tris-HCl buffered solution (0.4% w/v SDS, 100 mL) # Tris 18.2 g # SDS 0.40 g # distilled water 80 mL # 6 mol/L aqueous HCl for pH adjustment at pH 8.8 a proper quantity # distilled water for preparation of the 100 mL solution a proper quantity Sol. C : 0.50 mol/L Tris-HCl buffered solution (0.4% w/v SDS, 100 mL) # Tris 6.1 g # SDS 0.40 g # distilled water 90 mL # 6 mol/L aqueous HCl for pH adjustment at pH 6.8 a proper quantity # distilled water for preparation of the 100 mL solution a proper quantity Sol. D: 5.0 mmol/L Phos-tag TM AAL Solution containing 3% (v/v) MeOH # Phos-tag TM AAL-107 10 mg # methanol 0.10 mL # distilled water 3.2 mL The oily product, Phos-tag TM AAL-107 (10 mg) placed in a small plastic tube is completely dissolved in 0.10 mL methanol. The solution is diluted with 3.2 mL distilled water by pipetting. If a trace amount of insoluble material appeared as white fine powder (impurity) in the solution, it can be separated by centrifuging (2000 xg, 10 min) using two 2-mL microtubes. Store the solution in the 2-mL microtubes at 4˚C in the dark. From the supernatant solution, 45 mini-slab gels (50 µmol/L Phos-tag TM , 1-mm-thick, 9-cm-wide, 9-cm-long) can be prepared. Sol. E : 10 mmol/L MnCl 2 solution (50 mL, stored at room temperature) # MnCl 2 (H2O) 4 (FW. 197.9) 0.10 g # distilled water 50 mL The MnCl 2 solution is stable for at least 6 months. Do not use the other anion salt, such as Mn(NO 3)2. - 2 - Sol. F : 10 % w/v diammonium peroxydisulfate solution (0.30 mL) # (NH 4)2S2O8 (FW. 228.2) 30 mg # distilled water 0.30 mL The solution should be freshly prepared just before the acrylamide polymerization. Sol. G : Electrode buffer (0.5 L, pH is near 8.4, stored at room temperature) # Tris (25 mmol/L) 1.50 g # SDS 0.50 g # glycine (192 mmol/L) 7.2 g # distilled water 0.50 L Sol. H : Sample buffer 3x (10 mL, stored at –20˚C) # bromophenol blue (BPB, a tracking dye) 1.5 mg # SDS 0.60 g # glycerol 3.0 mL # Sol. C 3.9 mL # 2-mercaptoethanol 1.5 mL # distilled water for preparation of the 10 mL solution a proper quantity Sol. I : Acidic solution for fixation of proteins (1 L) # acetic acid 0.10 L # methanol 0.40 L # distilled water 0.50 L Sol. J : CBB staining solution (0.5 L) # Coomassie Brilliant Blue (CBB) 1.25 g # methanol 0.20 L # acetic acid 50 mL # distilled water 0.25 L After dissolving CBB in methanol, acetic acid and water are added into the solution. Sol. K : Washing and destaining solution (1 L) # methanol 0.25 L # acetic acid 0.10 L # distilled water 0.65 L Resolving gel solution (7 mL: e.g. , 100 µmol/L Phos-tag TM & 10% w/v acrylamide ) # Sol. A 2.33 mL # Sol. B 1.75 mL # Sol. D 0.14 mL # Sol. E (the same volume of Sol. D) 0.14 mL # distilled water 2.52 mL # TEMED (tetramethylethylenediamine) 20 µL # Sol. F 0.10 mL - 3 - Stacking gel solution (2 mL: e.g. , 4.5% w/v acrylamide ) # Sol. A 0.30 mL # Sol. C 0.50 mL # distilled water 1.08 mL # TEMED (tetramethylethylenediamine) 20 µL # Sol. F 0.10 mL 7. Casting Gels 1) Set up the casting apparatus ( e.g. , ATTO AE-6500 mini-slab gel system). 2) Prepare the resolving gel solution by mixing the solutions (see above, except for the catalysts Sol. F and TEMED). Degas the mixed solution less than 40 mmHg (0.05 atm) under stirring for 15 min. « See Troubleshooting 1 » 3) Add appropriate volume of Sol. F and TEMED (e.g., 50 µL and 10 µL, respectively) into the degassed solution and mix gently. The volumes must be adjusted for the Phos-tag electrophoresis. 4) Transfer the resolving gel solution (6.3 mL) between the plates, pore distilled water (1 mL) on top of the resolving gel solution, and then allow the gel to polymerize for ca. 30 min. 5) Stacking gel solution (see above) is prepared by a similar manner for the resolving gel. 6) Rinse the top of the resolving gel with distilled water and dry off the residual liquid with a paper towel. 7) Pore the stacking gel solution (1.8 mL) on top of the resolving gel and then insert the comb. 8) Allow the gel to polymerize for 1 h. 8. Sample Preparation 1) Mix sample with 3 µL Sol. H (+ a proper amount of distilled water) in a microcentrifuge tube and heat at 95 ˚C for 5 min (total volume of 9 µL). 2) Allow the solution to cool to room temperature. 3) Load the sample solution ( e.g. , 1.5 µL/well) using a micropipette. Phosphorylated proteins: # 1.8 mg/mL phosphorylated protein ( e.g., α-casein, β-casein, or ovalbumin) 1.0 µL # Sample buffer 3x (= Sol. H) 3.0 µL # distilled water 5.0 µL Dephosphorylated proteins: # 0.3 mg/mL dephosphorylated protein ( e.g., α-casein, β-casein, or ovalbumin) (alkaline phosphatase-treatmented proteins) 6.0 µL # Sample buffer 3x (= Sol. H) 3.0 µL Reagents for the dephosphorylation at 37 ˚C: # 10 mg/mL phosphorylated protein solution 50 µL # 0.50 M Tris-HCl buffer (pH 9.0) containing 0.10 M MgCl 2 10 µL # Sterilized water 39 µL # Alkaline phosphatase (Sigma-Aldrich) 0.33 unit # Reaction stop solution (= Sol. H) 3.0 µL/6.0 µL sample - 4 - 9. Electrophoresis 1) Assemble the electrophoresis equipments ( e.g. , ATTO AE-6500 mini-slab gel system) and fill the electrode chambers with the electrode buffer (= Sol. G). 2) Gently remove the comb from the stacking gel and load the samples into the wells using a micropipette. 3) Attach the leads to power supply ( e.g ., ATTO AE-8750 Power Station 1000XP). Run the gels under a constant current condition (30 mA/gel) until the BPB reaches the bottom of the resolving gel. « See Troubleshooting 2 » ATTO ATTO _ + 10. CBB Staining 1) Fix the proteins in the gel by soaking in Sol. I (50 mL) for ca. 10 min with gentle agitation.
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